Methods of monitoring fluid samples using multi-dimensional fluid sensors

ABSTRACT

The present invention provides multi-dimensional sensors with fluidic flow channels for processing fluid samples.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 12/994,302, which is a 35 USC 371 national phase application ofPCT/US2009/003550, with an international filing date of Jun. 12, 2009,which claims the benefit of priority to U.S. Provisional ApplicationSer. No. 61/073,865, filed Jun. 19, 2008, the contents of which arehereby incorporated by reference as if recited in full herein.

FIELD OF THE INVENTION

The present invention relates to sensors and automated analyzersthereof.

BACKGROUND

In the past, two-dimensional sensors, such as that shown in FIG. 1, havebeen used to analyze fluid samples. The two-dimensional sensors employ aflat platform with a counter electrode, reference electrode and workingelectrode. There remains a need to provide alternate sensorconfigurations, including, for example, those which can allow for one ormore of: multiple sample analysis, multiple testing on a sample, and/orparallel sampling, as well as automated or semi-automated analysisinstruments and system integration, for advancing low and high fluidsample throughput analysis, detection and/or diagnosis.

SUMMARY OF EMBODIMENTS OF THE INVENTION

Embodiments of the present invention are directed to multi-dimensionalsensors, as well as one or more of detectors, analyzers and methods ofgenerating, detecting and analyzing sensor data.

Some embodiments of the present invention are directed tomulti-dimensional fluidic sensor devices. The sensor devices include aplurality of sensors. Each sensor has a set of associated electrodes,including at least one working electrode, a reference electrode and acounter electrode. Each electrode in the set of electrodes is positionedone above another (e.g., substantially vertically arranged or stacked)and isolated by an electrical insulator therebetween. Each set ofelectrodes has aligned apertures that define at least a part of afluidic flow channel.

In particular embodiments, the working electrode has an inner wall thatsurrounds and defines the working electrode aperture, and at least aportion of the inner wall can optionally include a material selected toidentify a target analyte in a sample flowing through a respectivefluidic flow channel.

The target analyte may include a bioactive material of one or more ofthe following: an antibody, an antigen, a nucleic acid, a peptidenucleic acid, a ligand, a receptor, avidin, biotin, Protein A, ProteinG, Protein L, a substrate for an enzyme and any combination thereof.

In some embodiments, the multi-dimensional sensor has a top surface anda bottom surface with a plurality of apertures arranged in columns androws that form a plurality of fluidic flow channels. At least one set ofthe electrodes define at least a part of at least some of the pluralityof fluidic flow channels.

The plurality of fluidic flow channels may be configured as a pluralityof microfluidic flow channels.

Other embodiments are directed to three- or four-dimensional fluidicsensors. The sensor includes a sensor body having an array of fluidicflow channels formed therethrough. The fluidic flow channels include atleast one sensor that has: (a) at least one working electrode having anupwardly extending inner wall surrounding an aperture; (b) at least onecounter electrode having an upwardly extending inner wall surrounding anaperture and residing above or below the at least one working electrode;and (c) at least one reference electrode having an upwardly extendinginner wall surrounding an aperture and residing above or below the atleast one counter electrode. The working electrode aperture, the counterelectrode aperture and the reference electrode aperture are aligned todefine at least a portion of the fluidic flow channels.

The sensor can include an electrical insulator positioned between eachof the electrodes.

In some embodiments, the sensor body comprises a plurality of stackedlayers, including at least one working electrode layer, at least onereference electrode layer, and at least one counter electrode layer.Each layer has an array of apertures thereon configured and sized sothat, when aligned, the array of apertures of each of the layers defineat least a portion of the respective fluidic flow channels.

The layers can be sealed together, integral with each other, or snuglyattached to define discrete fluid-tight fluidic flow channels.

Still other embodiments are directed to methods of monitoring fluidsamples for detecting waterborne or airborne toxins or pathogens. Themethods include: (a) providing a sensor body having a plurality ofspaced apart fluidic flow channels, with the flow channels comprising atleast one sensor having a set of vertically stacked electrodes withaligned apertures that define at least a portion of the fluidic flowchannels; (b) flowing fluid samples through the fluidic flow channels;and (c) electronically detecting when a fluid sample tests positive fora selected analyte based on an output of the at least one sensor in arespective fluid channel.

The fluidic flow channels can optionally include a plurality ofdifferent sensors configured to detect different analytes. The flowingstep can be carried out to serially flow a respective fluid samplethrough a plurality of different fluidic flow channels in the sensorbody.

Yet other embodiments are directed to fluidic sensor devices. Thedevices include a sensor body having an array of microfluidic flowchannels, the sensor body having a plurality of stacked layers,including at least one working electrode layer, at least one referenceelectrode layer, and at least one counter electrode layer. Each layerhas a corresponding array of apertures thereon configured and sized sothat, when aligned, the array of apertures of each of the layers definesat least a portion of the fluidic flow channels.

Yet other embodiments are directed to fluidic detector systems foranalyzing fluid samples for target analytes. The detector systemsinclude: (a) a sensor body having a plurality of fluid flow channelsextending therethrough, the flow channels comprising a plurality ofsensors, each sensor having at least one upwardly extending workingelectrode with an aperture, wherein the respective sensor workingelectrode is in communication with counter and reference electrodes, thesensors configured to detect different selected analytes in a fluidsample; and (b) a sensor detector in communication with the sensor body,the sensor detector configured to electronically poll each sensor ineach flow channel individually to obtain a signal associated with apositive or negative test in response to the fluid sample passingthrough the fluidic flow channel.

Embodiments of this invention are directed to sensor bodies that can beassembled in a scalable configuration to include a selectable pluralityof 3-D sensor arrays to form 4-D sensor arrays with between about1-100,000 fluid channels, typically between about one and about 2000fluid channels, depending on sensor size.

It is noted that features of embodiments of the invention as describedherein may be methods, systems, computer programs or a combination ofsame although not specifically stated as such. The above and otherembodiments will be described further below.

Further features, advantages and details of the present invention willbe appreciated by those of ordinary skill in the art from a reading ofthe figures and the detailed description of the embodiments that follow,such description being merely illustrative of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a conventional (prior art)two-dimensional sensor array.

FIG. 2A is an isometric view of a multi-sample three or four dimensionalsensor array according to embodiments of the present invention.

FIG. 2B is a greatly enlarged schematic view of a stacked electrodearrangement associated with the sensor shown in FIG. 2A according toembodiments of the present invention.

FIG. 3 is a schematic illustration of a stacked electrode relationshipfor a sensor array similar to that shown in FIGS. 2A and 2B illustratinginsulating layers and reagents captured proximate a working electrodeaccording to embodiments of the present invention.

FIG. 4 is an isometric exploded view of a stacked counter, reference andworking electrode configuration for a sensor according to embodiments ofthe present invention.

FIG. 5 is an isometric view of a sensor array illustrating that multiplesets of the counter, reference and working electrodes shown in FIG. 4can be stacked to form a multi-test sensor according to embodiments ofthe present invention.

FIG. 6A is an isometric view of a multi-test, multi-sample (4D) sensorarray according to embodiments of the present invention.

FIG. 6B is an enlarged schematic view of the stacked relationship of thedifferent layers and electrode groups shown in FIG. 6A.

FIG. 7A is an isometric view of a multi-test, multi-sample (4D) sensorarray having a different electrode group arrangement according toembodiments of the present invention.

FIG. 7B is an exploded schematic illustration of the electrodearrangement shown in FIG. 7A.

FIG. 8A is an isometric view of a multi-test, multi-sample (4D) sensorarray similar to that shown in FIG. 7A, but with additional groups oflayers of the counter, reference and working electrodes according toembodiments of the present invention. FIG. 8B is an exploded view of theelectrode group configuration shown in the sensor array of FIG. 8A.

FIG. 9A is an isometric view of a multi-test, multi-sample (4D) sensorarray with columns of discrete sensors according to embodiments of thepresent invention.

FIG. 9B is an exploded view of the electrode group configuration shownin the sensor array of FIG. 9A, illustrating associated electricalcircuits that can be used to analyze the respective sensors in thesensor array according to embodiments of the present invention.

FIG. 10A is an isometric view of an electrical circuit interface thatcan be used to communicate with the sensors of the sensor arrayaccording to embodiments of the present invention.

FIG. 10B is a schematic illustration of three exemplary detector systemsthat can be used to interface with and/or detect the sensor data of thesensor arrays according to embodiments of the present invention.

FIG. 11 is a schematic illustration of an example of a test operationusing the stacked sensor array of embodiments of the present inventionfor an immunoassay to detect a target antigen according to embodimentsof the present invention.

FIG. 12 is a schematic illustration of an example of a test operationusing the stacked sensor array of embodiments of the present inventionfor a nucleic acid hybridization assay according to embodiments of thepresent invention.

FIG. 13 is a flow chart of operations that can be carried out to detectfluid (typically liquid) food-borne pathogens according to embodimentsof the present invention.

FIG. 14 is a flow chart of operations that can be carried out to detectairborne pathogens according to embodiments of the present invention.

FIGS. 15A and 15B are isometric views of a multi-dimensional sensorconfigured to serially flow a sample through more than one fluid channelaccording to embodiments of the present invention.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present invention is now described more fully hereinafter withreference to the accompanying drawings, in which embodiments of theinvention are shown. This invention may, however, be embodied in manydifferent forms and should not be construed as limited to theembodiments set forth herein; rather these embodiments are provided sothat this disclosure will be thorough and complete and will fully conveythe scope of the invention to those skilled in the art.

Like numbers refer to like elements throughout. In the figures, thethickness of certain lines, layers, components, elements or features maybe exaggerated for clarity. Where used, broken lines illustrate optionalfeatures or operations unless specified otherwise.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a,” “an” and “the” are intended toinclude plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises” or“comprising,” when used in this specification, specify the presence ofstated features, integers, steps, operations, elements components and/orgroups or combinations thereof, but do not preclude the presence oraddition of one or more other features, integers, steps, operations,elements, components and/or groups or combinations thereof.

As used herein, the term “and/or” includes any and all possiblecombinations or one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

Also as used herein, phrases such as “between X and Y” and “betweenabout X and Y” should be interpreted to include X and Y. Furthermore,phrases such as “between about X and Y” can mean “between about X andabout Y.” Also, phrases such as “from about X to Y” can mean “from aboutX to about Y.”

Further, the term “about” as used herein when referring to a measurablevalue such as an amount or numerical value describing any sample, flowrate, composition or agent of this invention, as well as any dose, time,temperature, and the like, is meant to encompass variations of ±20% orlower, such as, for example, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of thespecified amount.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the specification andclaims and should not be interpreted in an idealized or overly formalsense unless expressly so defined herein. Well-known functions orconstructions may not be described in detail for brevity and/or clarity.

It will be understood that when an element is referred to as being “on,”“attached” to, “connected” to, “coupled” with, “contacting,” etc.,another element, it can be directly on, attached to, connected to,coupled with and/or contacting the other element or intervening elementscan also be present. In contrast, when an element is referred to asbeing, for example, “directly on,” “directly attached” to “directlyconnected” to, “directly coupled” with or “directly contacting” anotherelement, there are no intervening elements present. It will also beappreciated by those of skill in the art that references to a structureor feature that is disposed “adjacent” another feature can includeportions that overlap or underlie the adjacent feature.

Spatially relative terms, such as “under,” “below,” “lower,” “over,”“upper” and the like, may be used herein for ease of description todescribe an element's or feature's relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if the device in thefigures is inverted, elements described as “under” or “beneath” otherelements or features would then be oriented “over” the other elements orfeatures. Thus the exemplary term “under” can encompass both anorientation of over and under. The device may otherwise be oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein interpreted accordingly. Similarly, the terms“upwardly,” “downwardly,” “vertical,” “horizontal” and the like are usedherein for the purpose of explanation only, unless specificallyindicated otherwise.

It will be understood that, although the terms first, second, etc., maybe used herein to describe various elements, components, regions, layersand/or sections, these elements, components, regions, layers and/orsections should not be limited by these terms. Rather, these terms areonly used to distinguish one element, component, region, layer and/orsection, from another element, component, region, layer and/or section.Thus, a first element, component, region, layer or section discussedherein could be termed a second element, component, region, layer orsection without departing from the teachings of the present invention.The sequence of operations (or steps) is not limited to the orderpresented in the claims or figures unless specifically indicatedotherwise.

The term “sensor” refers to a device having one or more electrodes thatcan include analytical sites arranged on and/or in one or moresubstrates that permit one or more analyses to be performed on one ormore fluid samples (e.g., microsamples) at the same time and/or atdifferent times, typically, but not limited to, via flowable throughputthrough fluidic channels in the device. The fluid test sample can be inor comprise substantially gas or liquid. The test sample may includesolid or particulate matter in the fluid. The flowable throughput may,in some embodiments, be high throughput conditions at a rapid flowrate(s). Flow speed can range from about 1 μl per minute for a simpleflow-through assay (e.g., a sample passes through the fluid channelrelatively slowly and no incubation is needed) to about 10 ml per minute(or more) for some tests or assays. The term “3D” or “three-dimensional”sensor or sensor array refers to a sensor with a stacked (one overanother) electrode arrangement. The term “sensor array” means that thedevice has more than one sensor, typically arranged in a repeating orpartially repeating pattern or layout on one or more surfaces. The term“4D” or “four-dimensional” sensor or sensor array refers to a sensordevice that includes multiple sensors in a respective fluid channel thatcan carry out multiple tests per sample and/or analyze multiple samples,serially and/or in parallel. The multi-dimensional sensor arrayscontemplated by embodiments of the present invention can be configuredto concurrently accept and test multiple different samples and performmultiple different analyses on those samples and/or serially test asingle sample or a plurality of samples.

A “fluidic flow channel” refers to a continuous or uninterrupted fluidpathway or channel typically extending through the sensor array, andtypically with an opening at either an outside edge, an end or top orbottom of the sensor array (i.e., an inlet and an outlet) to allow thepassage of fluid therethrough, from a sample entry location to a sampledischarge location. The device can be configured to re-circulate or flowthe fluid sample through one or more sensor channels over time, such asby using different fluid delivery systems, including, for example,pumps, vacuums or capillaries. A “microfluidic” flow channel is aminiaturized fluidic flow channel that accommodates a small fluidvolume, typically between microliters and nanoliters of fluid. Themicrofluidic flow channel typically can hold or accommodate microscaleamounts, e.g., microliters or less, such as, for example, 100microliters or less, including nanoliters of fluid, which can be in theform of a gas or liquid as noted above. In some particular embodiments,each channel can, for example, hold from a sub-microliter volume (e.g.,about 0.1 μl) to about a 100 μl volume. In some embodiments, forexample, a channel can hold between about a 1 μl volume to about a 10 μlvolume. For example, if one channel holds about 2 μl of liquid, a sensorwith 20 channels can process about 40 μl of sample to test for 20analytes.

Embodiments of the invention may be particularly suitable for lab orfield testing of water systems, terrestrial or extraterritorialenvironments or fluids. For example, embodiments of the presentinvention may be used to monitor commodities or environments that may besubject to a security and/or health risk, e.g., air sampling, samplingof water systems including water treatment systems, and sampling ofcomponents or environments in food industries such as food productionsystems.

The sensor arrays of the present invention can be configured into anysuitable geometric shape that defines the multiple sensor analysissites. In some embodiments, the sensor arrays are configured asmulti-layer cubes. The term “cube” is not limited to a “cube” shape, butis used broadly to refer to a box-like shape, such as a substantiallyrectangular shape or cube shape. However, the sensor arrays may have anydesired geometric shape, and are not required to have a straight edge.

The term “bioactive” includes the term “bioreactive” and means an agentor material or composition that alone or when combined with anotheragent and exposed to a test sample will undergo a chemical or biologicalreaction and/or be altered in appearance or in another optically orelectronically readable or detectable manner when a target analyte,e.g., constituent, antigen, antibody, bacterium, virus, ligand, proteincontaminant, toxin, radioactive material and/or other material ispresent in the test sample. See, e.g., U.S. Pat. No. 6,294,107, thecontents of which are hereby incorporated by reference as if recited infull herein.

The term “insulator” refers to a material that can provide electricalinsulation between one or more adjacent components, e.g., between acounter and reference electrode and/or between a reference and workingelectrode. The insulator may also be able to provide fluid isolationbetween stacked layers. In other embodiments, two or more insulatorlayers may be used: at least one for electrical isolation and at leastanother one for fluid sealant. The fluid sealant material can cooperatewith adjacent layers to define a substantially fluid-tight seal. Thefluid sealant may be a thin gasket layer of any suitable material, suchas, for example, a polymer, rubber, and/or metal. In some embodiments,the fluid sealant can be integrated into the electrical insulator and/orlaminated and/or otherwise attached thereto. Where gaskets are used, thegasket may have a thickness that is substantially the same or more orless than an adjacent electrode layer, and is typically thinner than atleast the working electrode layer. In some embodiments, the gasket canbe formed of an elastically compressible material. In some embodiments,the fluid sealant can comprise a gasket of thermoplastic elastomers(including but not limited to Viton®, Buna-N, EPDM, and Versaflex®materials) and/or silicone rubbers.

Turning now to the figures, FIG. 2A illustrates a sensor array 10 witheach sensor 20 having an associated (typically substantiallyvertically-stacked) group of electrodes 25, including a workingelectrode 30, a reference electrode 40, and a counter electrode 50, oneabove another. As shown in FIG. 2B, an electrical insulator 55 canreside between the working electrode 30, the reference electrode 40, andthe counter electrode 50. Although the sensor electrode groups 25 areshown in a tubular form in an exploded view such as in FIG. 2B (and FIG.3, etc.), this shape is merely for ease of discussion. The sensorelectrode groups 25 are configured with aligned apertures that definefluid flow channels 60 as shown in FIG. 2A.

FIG. 3 illustrates a different arrangement of the electrode group 25 andalso illustrates that the working electrode 30 includes a coating 33 oris embedded or otherwise provided with a material that provides thedesired test analyte for a respective sensor 20 for the test detectionand/or monitoring. The coating 33 can comprise any suitable materialsuch as, for example, capture reagents or any suitable bioreactivematerial.

FIG. 3 also illustrates that the insulator 55 both electricallyinsulates and provides a fluid seal between the adjacent layers, atleast upon assembly. That is, the entire stacked configuration can becompressed together and the insulator 55 defines the fluid seal.Alternately, the fluid seal can exist upon assembly of the adjacentlayers, such as by size and configuration or attachment means, includingadhesive, brazing, welding and the like. Examples of suitable insulatormaterials include, for example, silicone rubber and certain thermoelastomers such as, for example, Versaflex®, and can, in someembodiments, have thicknesses ranging from between about 0.05 mm toabout 10.0 mm. Different insulator materials can be used for differentlayers (or even partial layers).

FIGS. 2A and 3 illustrate that the sensor array 10 can include an arrayof fluid flow channels 60, having from n=1 to n=Y columns and from n=1to n=X rows. Similarly, the flow channels can have from n=3 primarylayers to n=Z layers to scale the sensor array in any desiredconfiguration for the desired application. Each channel 60 may define aseparate test path which can expose the test sample to a plurality ofdifferent test sites 20 at different levels of the working electrodes 30in the flow channel 60.

In some embodiments, each electrode group 25 can define a discretesensor 20 performing a different test, or the same test for reliability,and/or redundancy. Each working electrode 30 can comprise a differentmaterial 33, the same material, or even a different concentration orformulation of the same material for sensitivity or specificity ofconcentration or the like. Hence, a sensor array 10 can carry out anumber of different tests e.g., tests n=1, to n, where “n” is any numberbetween 1 and 500,000, typically, less than 100,000, and in someembodiments between about two to about 3000. As shown in FIG. 2A, eachchannel 60 can perform one test and in FIG. 3, the lower figure of thesensor array 10 illustrates three tests (e.g., three working electrodes30) while the schematic illustrates six tests (e.g., six workingelectrodes 30). Also, each channel 60 residing in fluid communication inan X-Y location (row and column) of the sensor array 10 can define adifferent sample flow channel, allowing for a relatively large number oftest samples to pass through the sensor array 10 or for one sample to betested in the different samples over time. Thus, for example, in FIG.2A, the sensor array 10 has 10 rows, X=10, 8 columns, Y=8, and onesensor in each channel 60, Z=1, thus, there are 80 tests 65 available inthis sensor array 10 and up to 80 samples can be accommodated by thissensor array 10. FIG. 6A illustrates tests 65 ₁-65 _(n). The bottom ofFIG. 3 illustrates an example of a 4-D sensor array 10, where Z=3 (3working electrodes 30 in each channel 60), eight rows, X=8, and 6columns, Y=6. This sensor array 10 can accommodate up to 48 samples andperform up to 144 different tests (8 rows, 6 column and 3 sites percolumn, 8×6×3). The sensor array 10′ can be configured to carry out anumber of tests in a respective channel 60, typically between 1-1000,more typically between 1-50, and the sensor array 10′ can evaluate oneor more samples, such as, for example, but not limited to, up to about100,000 samples, typically up to about 2000 samples, for each of thedifferent tests (each test can be carried out by a corresponding sensor20). It is noted that each working group of electrodes 25 that defines arespective sensor 20 can be configured to share one or more referenceelectrodes 40 and may include one or more working electrodes 30.

FIG. 4 is an exploded view of an exemplary 3-D sensor array 10. Asshown, the sensor array 10 has a plurality of sensors 20 with each ofthe electrode groups 25 being defined by layers of adjacent materialsthat form the respective electrodes 30, 40, 50. The working electrodelayer 30L, the reference electrode layer 40L and the counter electrodelayer 50L are closely spaced and separated by a relatively thininsulation layers 55. Each of the layers 30L, 40L, 50L and 55L hascorresponding patterns or arrays of apertures 31, 41, 51 and 56, that,when aligned and assembled, define the fluid channels 60. The layers30L, 40L, 50L can be formed sequentially, one over the other, or may belaminated or otherwise held snugly together to define the fluidic flowchannels 60. Thus, the layers may be integral with each other orseparate or combinations of same. In particular embodiments, the workingelectrode layer 30 can have a thickness that is between about 0.05 mm toabout 12 mm. The counter electrode 50 can comprise insert materials,such as noble metals or graphic carbon to avoid dissolution. Commonlyused reference electrodes 40 include silver/silver-chloride electrodes,calomel electrodes, and hydrogen electrodes. The surface of a workingelectrode 30 is typically where the biochemical reactions take place.Besides behaving as an electrode for electroanalysis, the capturebiomolecules, such as proteins, antibodies, antigens, or DNA probes, canbe coated or otherwise disposed on the surface of the working electrode30. The surface chemical properties of a working electrode 30 can varydepending on applications. For coating proteins on a working electrode30, for example, the surface can be plated with a thin layer of gold.

The working electrode layer 30 can comprise an analyte 33 forfacilitating the testing and the sensor array 10 can define a singletest 65 ₁ for multiple samples, or multiple tests for one sample overtime (by directing the sample 63 through another different channel 60(serially) over time, such as shown by the arrows and fluid flow ofsample 63 in FIGS. 15A, 15B). The analyte 33 can be placed on a wall orportion of a wall of each working electrode 30 w forming a portion ofthe flow channel 60. The analyte 33 can be, for example, molded into oronto (e.g., alone or via a matrix), coated, sprayed, injected, poured,painted, covered, impregnated, vapor deposited, permeated, plated,soaked and/or embedded with an analyte or analytes 33. The material 33can also be applied by a shrink-wrap or adhesively attachable strip orpatch to or otherwise applied to an entire or partial wall of theworking electrode 30. In some embodiments, each layer 30L has the sameanalyte 33. However, the invention is not limited thereto as differentanalytes 33 may be applied to different portions of the layer 30L.

In some embodiments, a first analyte such as a first bioactive agent ormaterial can be present on a first portion of the wall 30 w and a secondanalyte such as a second bioactive agent or material can be present onthe same working electrode (not shown). In certain embodiments, thelayer 30L can be immersed or soaked in a solution comprising the analyte33, e.g., bioactive agent or material, resulting in the presence of thebioactive agent or material on both upper and lower (top and bottom)surfaces of the layer 30L, as well as on the wall surfaces 30W formingthe apertures 31.

FIG. 5 illustrates that different selected 3-D sensor arrays 10 can beassembled together to form a 4-D sensor array 10′. In the embodiment,each 3-D sensor array 10 is shown as configured the same for ease ofassembly. Also, in this embodiment, three sensor arrays 10 are stackedone above another to form three tests, 65 ₁, 65 ₂, 65 ₃, that can becarried out on a test sample in a respective channel 60 substantiallysimultaneously. These sensor arrays 10 can be pre-assembled and providedto the lab or field test agency or may be selected onsite for aparticular application. As such, the sensor arrays 10 can be supplied inkits of different sets of tests or ordered separately for subsequentassembly and use. Each three-dimensional sensor array 10 can have thesame or a different material 33 for use in the four-dimensional sensorarray 10′. Typically, once assembled, the sensors 10, 10′ do not need tobe disassembled to be analyzed or monitored as will be discussed furtherbelow.

FIGS. 6A and 6B illustrate a four-dimensional biosensor 10′ with themultiple different 3-D sensors 10 assembled together to form 65 ₁-65_(n) (shown as 5 different) tests per sample. The exemplary sensor array10′ shown can accommodate up to 88 samples (if some channels 60 are notused to retest or re-circulate one or more samples).

FIGS. 7A and 7B illustrate that the sensor arrays 10 can be formed indifferent electrode layer configurations. As shown, each sensor 20′includes a plurality of working electrodes 30 with a shared counterelectrode 50 s and a shared reference electrode 40 s. In someembodiments, an increased number of working electrodes 30 per sensor 20′(e.g., test 65 ₁) with a potential decrease in their size may reduceelectrical noise in one or more of the sensors 20′.

FIGS. 8A and 8B illustrate the sensor configuration 20′ of FIGS. 7A and7B in a 4-D sensor array 10′. Combinations of the sensor configurations20′ shown in FIGS. 7A and 7B as well as those shown in earlier or laterfigures may also be used as well as different numbers of workingelectrodes 30 and shared or dedicated reference and counter electrodeconfigurations 40, 50, respectively.

FIG. 9A illustrates an exemplary 4-D sensor array 10′. In thisembodiment, the different sensors 20 share a counter electrode 50. FIG.9B illustrates an exemplary circuit diagram of an interface circuit 90with electronic connections to individual sensors 20. This circuit ismodified where more than one counter electrode 50 is used as will beappreciated by one of skill in the art. The columns of sensors 20defining respective channels 60 can be selected and switchedelectronically for activation, evaluation, monitoring detection and/orthe like. In operation, the sensors 20 can be activated to becomeoperational to carry out the fluid sampling. In other embodiments, thesensor 20 may be passive and only activated for detection or reading.The sensors 20 may be individually activated or a column or all of thesensors 20 may be concurrently or serially activated in a common flowchannel 60. Each sensor 20 can be individually detected using a systemdetector that electronically communicates with the sensor electronics.Typically, the sensors 20 in a respective sample well/channel 60 areactivated together. The sensors 20 can be read substantially with theactivation or at a later time. Each sensor 20 in a channel 60 and/or inmore than one channel 60 can be individually or serially read ormonitored or read or monitored at the same time. Thus, the detection ormonitoring of the sensors 20 can be done individually, serially and/orconcurrently, allowing the sensors 20 to be polled in any desirablearrangement. In some embodiments, a review protocol can be used to“triage” the sensors 20 and identify those with increased strength ofsignal which can be read first.

The various electrode layers 30L, 40L, 50L can have electrical pathsthat extend to an outside perimeter of the sensor array 10, 10′ to allowfor each sensor to be (individually) activated and/or detected. Theelectrical paths in the various layers 30L, 40L, 50L be formed usingvias or other paths (see, e.g., FIG. 9B).

FIG. 10A illustrates an electronic interface 100 that can provide theelectrical circuit 90 to connect to the sensor array 10, 10′ (shown as10′). The interface 100 can include a polymer or other suitable casesandwiching alternating conductive/non-conductive layers 92, 93extending between the sensor array 10′ and the contacts on the interface(e.g., PCB) and in communication with the various electrode layers 30L,40L, 50L associated with each sensor 20 in each channel 60.

FIG. 10B schematically illustrates a sensor array 10, 10′ incommunication with fluid samples 70, and a fluid handling system 88 thatcan communicate with different exemplary sensor detectors 500 that cancommunicate with the sensor arrays 10, 10′ and can extract test datatherefrom. The fluidics assembly 88 can be configured to releasably holdsensor arrays 10, 10′ of various heights; as such they may vary in usedepending on the number of stacked 3-D sensor arrays 10′ are usedtest-to-test or user-to-user (where the sensor arrays 10′ are made fromselectively attachable sensor arrays 10). As shown, in some embodiments,the fluidics assembly 88 resides in fluid communication with one or moreof the flow channels 60 at an upper surface of the sensor array 10, 10′.Other fluid delivery/flow systems and configurations may also be used.

One exemplary detector 500 is an electrochemical detector 510. Theelectrochemical detector 510 reads electrochemical signals generated bythe sensors 20 in the sensor array 10, 10′. The sensors 20 convertchemical signals to electrical signals and those signals are relayed ortransmitted to external electrical contacts using an interface 90 withan array of conductors. The electrochemical detectors 510 can includede-multiplexers, amplifiers and A/D converters, filters and the like, asis known to those of skill in the art.

Another exemplary detector 500 is an optical detector 520 that comprisesa light source, such as a laser 522, that can transmit a light into asensor space to interrogate the sensors 20, and a light sensor 525 incommunication with the sensor array 10, 10′ and laser to be able toreceive transmitted light in response thereto. In this embodiment, thesensors 20 are configured to optically change in opacity, color,intensity, transmissiveness, or the like, which can be opticallydetected. For example, sensors having fluorescent or chemiluminescentproperties are examples of optical sensors. A sensing element or groupof elements (e.g., working electrode 30) can be illuminated or excitedand their light intensity can be converted to an electrical signalexternally by using, for example, a PMT (photomultiplier tube). Thedetector 520 can include mirrors, lenses and other optical componentssuitable for optical detection as is known to those of skill in the art.

FIG. 10B also illustrates a third type of detector 500, a magneticresonance detector 530. In this embodiment, magnetic labels can beattached to the sensor sites as detection probes or elements. Thesemagnetic labels can be quantified or assessed by measuring perturbationsof an applied external magnetic field 535 that extends proximate thesensor array 10, 10′.

FIGS. 11 and 12 illustrate exemplary test targets that can be analyzedaccording to particular embodiments of the present invention. As shown,FIG. 11 illustrates an immunoassay test with multiple test sites 65 ₁-65n, and with the sensor 20, e.g., the working electrode 30 having ananalyte 33 with a detection antibody with HRP. FIG. 12 illustrates anexample of a DNA hybridization assay where the test material 33 onand/or in the sensor 20, e.g., working electrode 30 includes a targetDNA probe.

FIG. 13 is a flow chart of operations that can be used to carry outembodiments of the present invention. This flow chart is directed tomonitoring for and/or detecting fluid borne pathogens in food or otherconsumer consumable items in a food production facility. At least onemulti-dimensional sensor array 10, 10′ can be placed in fluidcommunication with a production line (e.g., bottling or packaging stagein a production line)(block 200). One or more samples from different ormultiple stages of the production line can be obtained (block 210). Thesamples can be captured, washed, filtered or processed (pre-processed)as appropriate, and flowed through one or more of the channels 60 in thesensor array 10, 10′. One or more sensors 20 in the array 10, 10′, suchas one or more in a flow channel 60, can be read (block 230) by adetector and an alert generated locally and/or to a command and controlcenter (block 235) if a reading is positive. The command and controlcenter can be in communication with a regulatory agency, such as, forexample, the United States Food and Drug Administration or HomelandSecurity Office. Another sensor 20 in the array 10, 10′, typically at adifferent level and/or testing for a different analyte, can be read(block 240). Another sample can be introduced into the sensor array 10,10′ (block 242). Maintenance of the sensor array 10, 10′ can bescheduled for desired intervals, such as daily, weekly, monthly and thelike (block 250). A new sensor array 10, 10′ can be installed or theexisting sensor array 10, 10′ can be retrofitted with one or morecomponents. The sensor arrays 10, 10′ can be configured with modularcomponents that allow for ease of repair/upgrade. Examples ofrefurbishment components include, for example, fresh buffers,insulators, fluid seals and the like. Both positive and negativecontrols can be installed in the sensor array 10, 10′, or one array 10,10′ can include the positive controls and another the negative controls,for monitoring system performance assessment and reliability testing(block 255). For ease of assessment, a false positive control sensor maybe adjacently positioned next to its “normal” sensor, although otherplacements in the sensor array 10, 10′ or in a different sensor orsensor array are also possible.

FIG. 14 is a flow chart of operations that can be used to carry outembodiments of the present invention. This flow chart is directed tomonitoring for and/or detecting airborne pathogens. An air samplerobtains samples of air (block 300). The air is introduced into thesensor array 10, 10′ (block 320). The air may optionally be liquefiedinto a flowable sample prior to step 320 (block 310). At least onemulti-dimensional sensor array 10, 10′ can be placed in fluidcommunication with a production line (e.g., bottling or packaging stagein a production line)(block 200). The air samples can be captured,washed, filtered or processed (pre-processed) as appropriate, and flowedthrough one or more of the channels 60 in the sensor array 10, 10′. Oneor more sensors 20 in the array 10, 10′, such as one or more in a flowchannel 60, can be read (block 330) by a detector and an alert generatedlocally and/or transmitted to a command and control center (block 335)if a reading is positive. The command and control center can be incommunication with a regulatory agency, such as, for example, the UnitedStates Food and Drug Administration or Homeland Security Office. Anothersensor 20 in the array 10, 10′, typically at a different level and/ortesting for a different analyte, can be read (block 340). Another samplecan be introduced into the sensor array 10, 10′ (block 342) (after orwith the first sample). Maintenance of the sensor array 10, 10′ can bescheduled for desired intervals, such as daily, weekly, monthly and thelike (block 350). A new sensor array 10, 10′ can be installed or theexisting sensor array 10, 10′ can be retrofitted with one or morecomponents. The sensor arrays 10, 10′ can be configured with modularcomponents that allow for ease of repair/upgrade. Examples ofrefurbishment components include, for example, fresh buffers,insulators, fluid seals and the like. Both positive and negativecontrols can be installed in the sensor array 10, 10′ or one array 10,10′ can include the positive controls and another the negative controls,for monitoring system performance assessment and reliability testing(block 355). That is, to assess “false positives” or a control sensormay be used in communication with another sensor that is configured torender a regular positive report for a processed sample. For ease ofassessment, a false positive control sensor may be adjacently positionednext to its “normal” sensor, although other placements in the sensorarray 10, 10′ or in a different sensor or sensor array are alsopossible.

FIGS. 15A and 15B illustrate that a single sample 63 can be routedthrough more than one channel 60. FIG. 15A illustrates that the sampletravels from a bottom of a first channel 60 up through the top and backinto the top of an adjacent or closely spaced channel 60. FIG. 15Billustrates that the sample 63 enters a first channel from the bottom,exits the top, and is rerouted to enter a bottom of another channel 60.In some embodiments, a single sample can be routed through each channel60 of substantially the entire array 10, 10′, or each channel in a rowor a column of the array. The fluid delivery system (e.g., manifold) canbe connected to take a discharged sample and reroute it into anotherchannel 60. The sample entry for a respective channel 60 can be all fromthe top, all from the bottom or alternately from the top to the bottom,then bottom to top.

The sensor arrays 10, 10′ can have a surface comprising predeterminedelectronically and/or optically readable indicia 600 as shown in FIG.15B. Such indicia 600 can be placed on or in the sensor array 10, 10′during manufacture, or such indicia 600 can be placed after manufacturein the form of bar code, color code, symbols, watermark, icons, and/or amicrochip with a secure “electronic handshake” or interface thatcommunicates with an automated reader or analyzer. The location of theindicia 600 may be such that it is not readily visually apparent by thenaked eye, and may be varied sensor to sensor 10, 10′. The location ofthe indicia 600 may be electronically correlated via a batch ormanufacturer code or the like. The indicia 600 can be in any form or inmultiple forms for redundancy, e.g., a bar code, a sticker, plate,notch, etching, etc. These indicia 600 can be used, for example, toidentify the sensor array 10, 10′ and/or other characteristics of thetests thereon (e.g., order or position of each sensor 20 in the stack,identification of bioactive agent(s) or material(s) present on theworking electrode, status of testing of samples and/or analyzing ofsignal, etc.) and/or to verify the authenticity of the sensor array 10,10′. These indicia 600 can be placed in any location (e.g., top, bottom,edge, under a gasket, on a gasket, or on a test surface 30 w) and canalso be present at multiple locations on the same array 10, 10′.

The indicia 600 can be visually, optically and/or electronicallyreadable at the initiation of a test and/or before assembly of thesensors 10 to verify the type of test thereon and/or the authenticity ofthe chip to help control counterfeit products and/or inaccurate testing.For example, the electronic detector or reader 500 (see, e.g., FIG. 10B)can interrogate the sensor arrays 10, 10′ and identify whether thesensor array is authorized or authentic. The reader can also beconfigured to alert a user when an unauthorized sensor array is detectedand may even be programmed to block an analysis of such a sensor orprominently disclaim the test results where such authenticity isquestioned. This may allow a clinician or laboratory technician or otheruser to retest a sample or investigate the test results rather than relyon potentially false test analysis.

Further embodiments of this invention include an automated method ofanalyzing multiple samples exposed to multiple analytical sites in abiosensor array, comprising: a) introducing a multiplicity of fluidsamples into a fluid delivery system of an automated bioanalyzer; and b)flowing the multiplicity of fluid samples through the biosensor arrayhaving sets of electrodes defining at least one sensor with aperturesdefining microfluidic flow channels. An inner wall of at least oneelectrode in fluid communication with at least one of the flow channels.The electrode wall can include at least one bioactive agent or materialthat contacts a sample flowing thereover. An analyzer can analyzesignals obtained from the sensors.

Non-limiting examples of a bioactive agent or material of this inventioninclude an antibody, an antigen, a nucleic acid, a peptide nucleic acid,a ligand, a receptor, avidin, streptavidin, biotin, Protein A, ProteinG, Protein L, a substrate for an enzyme, an anti-antibody, a toxin, apeptide, an oligonucleotide and any combination thereof.

The bioactive agent or material can be attached directly to the sensor,e.g., an inner wall of the working electrode and/or the bioactive agentor material can be attached indirectly (i.e., via a linker such as PEG(polyethylene glycol), EDC (N-3-Dimethylaminopropyl-N′-ethylcarbodiimidehydrochloride), glutaraldehyde, etc.). The bioactive agent can also beattached through a mediate layer of biotin, avidin, polylysine, BSA(bovine serum albumin), etc. as is known in the art. The bioactive agentor material of this invention can also be provided to an analytical sitein a fluid solution, e.g., in order to detect a reaction at theanalytical site.

In some embodiments, the bioactive material can be an antibody orantibody fragment and a signal is detected if an antigen/antibodycomplex is formed. In such embodiments, as an example, a first antibodyor antibody fragment can be attached directly or indirectly to a wall orsurface of the sensor via any variety of attachment protocols standardin the art. Then a fluid test sample is passed through a microfluidicflow channel such that the sample contacts an analytical site thatcomprises the immobilized first antibody or antibody fragment. If thereis an antigen in the test sample that is specific for the immobilizedfirst antibody or antibody fragment, the antigen will be bound (i.e.,“captured”) by the immobilized first antibody or antibody fragment,resulting in the formation of an antigen/antibody complex immobilized onthe sensor. A fluid comprising a second antibody or antibody fragmentthat is detectably labeled is then passed through the microfluidic flowchannel. The detectably labeled second antibody or antibody fragment isalso specific for the antigen bound by the first immobilized antibodyand will therefore bind to the captured antigen, thereby immobilizingthe detectably labeled second antibody or antibody fragment at theanalytical site. Upon subsequent analysis, the immobilized detectablylabeled second antibody is detected at the analytical site according tothe methods described herein and as are well known in the art for suchdetection. The result of the analytical testing is that the test samplecomprises (e.g., is positive for) the target antigen.

In some embodiments, the bioactive material can be an antigen and asignal is detected if an antigen/antibody complex is formed. In suchembodiments, as an example, an antigen (e.g., a peptide, polypeptide,amino acid sequence defining an epitope, etc.) is attached directly orindirectly to a surface of the sensor(s) via any variety of attachmentprotocols standard in the art. Then a fluid test sample is passedthrough a microfluidic flow channel such that the sample contacts ananalytical site that comprises the immobilized antigen. If there is anantibody in the test sample that is specific for the immobilizedantigen, the antibody in the sample will be bound (i.e., “captured”) bythe immobilized antigen, resulting in formation of an antigen/antibodycomplex immobilized on the sensor (e.g., working electrode wall). Afluid comprising a detectably labeled anti-antibody or antibody fragmentspecific for an antibody of the species from which the test sample wasobtained is then passed through the microfluidic flow channel. Thedetectably labeled antibody or antibody fragment will bind theimmobilized antibody captured by the antigen, thereby immobilizing thedetectably labeled antibody or antibody fragment at the analytical site.Upon analysis, the immobilized detectably labeled antibody is detectedat the analytical site according to the methods described herein and asare well known in the art for such detection. The result of theanalytical testing is that the test sample comprises (e.g., is positivefor) the target antibody.

In other embodiments, the bioactive material can be a nucleic acid orpeptide nucleic acid and a signal is detected if a nucleic acidhybridization complex is formed. In such embodiments, as an example, anucleic acid (e.g., an oligonucleotide) or peptide nucleic acid (PNA) isattached directly or indirectly to a surface of the sensor(s) via anyvariety of attachment protocols standard in the art. Then a fluid testsample is passed through a microfluidic flow channel such that thesample contacts an analytical site that comprises the immobilizednucleic acid or PNA. If there is a nucleic acid in the test sample thatis complementary [either fully complementary or of sufficient partialcomplementarity to form a hybridization complex under the conditions ofthe assay (e.g., high stringency, medium stringency or low stringency assuch terms are known in the art)], the nucleic acid in the sample willhybridize to (i.e., “be captured by”) the immobilized nucleic acid orPNA, resulting in formation of a hybridization complex immobilized onthe sensor. Upon (subsequent) analysis, the immobilized hybridizationcomplex is detected at the analytical site according to the methodsdescribed herein and as are well known in the art for such detection.The result of the analytical testing is that the test sample comprises(e.g., is positive for) the target nucleic acid. In some embodiments,the immobilized hybridization complex can be detected because thenucleic acid in the test sample has been modified to comprise adetectable signal (e.g., fluorescence, chemiluminescence, radioactivity,electrochemical detection, enzymatic detection, magnetic detection, massspectroscopy etc.).

The examples set forth above describing various assays that can becarried out in the sensors of this invention are not intended to belimiting in any way. If a target analyte can be captured by acorresponding bioactive agent that can be attached to the sensor, andthe analyte can be detected by one of the detection methods listed aboveor other methods, then the assay can be performed using the sensorsaccording to embodiments of this invention. The sensors can be employedto carry out any type of direct immunoassay, indirect immunoassay,competitive binding assay, neutralization assay, diagnostic assay,and/or biochemical assay. For example, a prenatal and/or neonatal TORCHassay, antigens and/or antibodies specific to toxoplasmosis, rubella,cytomegalovirus and herpes simplex virus can be attached on the sensorsfor capturing both IgG and IgM antibodies and/or viral antigenscorresponding to the pathogens in human serum. As another example,antibodies and/or antigens specific to human Hepatitis B and C can beattached for detecting antibodies specific to surface and core antigensof the virus and/or the antigens in human serum samples. Anotherexample, a substrate is immobilized on the sensor (e.g., inner wall ofthe working electrode) and a fluid sample is passed over the immobilizedsubstrate to detect an enzyme that specifically acts on the immobilizedsubstrate. A product of such enzyme activity can be detected, resultingin the identification of a test sample positive for the target enzyme.

Non-limiting examples of pathogens, agents of interest and/orcontaminants that can be detected, identified and/or quantitatedaccording to methods and devices of embodiments of the inventionsinclude a majority of pathogens causing infectious diseases in human andanimal, food and air borne pathogens, and pathogens which can be used asbioterrorism agents. The sensors can also be used to detect antibodiesand proteins which can be used to diagnose a majority of infectiousdiseases and other diseases and conditions (e.g. thyroid function,pregnancy, cancers, cardiac disorders, autoimmune diseases, allergy,therapeutic drug monitoring, drug abuse tests, etc.). It would be wellunderstood to one of ordinary skill in the art that the methods andsensors according to embodiments of this invention can also be employedto detect, identify and/or quantitate specific nucleic acids in a sample(e.g., mutations such as insertions, deletions, substitutions,rearrangements, etc., as well as allelic variants (e.g., singlenucleotide polymorphisms). Nucleic acid based assays of embodiments ofthis invention can also be employed as diagnostics (e.g., to detectnucleic acid of a pathogen in a sample). In some embodiments, mutationsof cytochrome P450 genes and blood clotting factor genes can be detectedand/or identified. The sensors of embodiments of this invention can alsobe used to determine the level of a RNA transcript by hybridizing alabeled complex mixture of RNA samples onto surfaces coated withcomplementary strands of oligonucleotides or cDNAc.

The foregoing is illustrative of the present invention and is not to beconstrued as limiting thereof. Although a few exemplary embodiments ofthis invention have been described, those skilled in the art willreadily appreciate that many modifications are possible in the exemplaryembodiments without materially departing from the novel teachings andadvantages of this invention. Accordingly, all such modifications areintended to be included within the scope of this invention as defined inthe claims. The invention is defined by the following claims, withequivalents of the claims to be included therein.

What is claimed is:
 1. A method of monitoring fluid samples fordetecting waterborne or airborne toxins or pathogens, comprising:providing a sensor body having a plurality of spaced apart fluidic flowchannels, with the flow channels comprising at least one sensor having aset of vertically stacked electrodes with aligned apertures that defineat least a portion of the fluidic flow channels; flowing fluid samplesthrough the fluidic flow channels; and electronically detecting when afluid sample tests positive for a selected analyte based on an output ofthe at least one sensor in a respective fluid channel.
 2. The method ofclaim 1, wherein the fluidic flow channels comprise a plurality ofdifferent sensors configured to test for different pathogens or toxins,and wherein the flowing step comprises serially flowing a respectivefluid sample through a plurality of different fluidic flow channels inthe sensor body.
 3. The method of claim 1, wherein the different sensorsare configured to test for different pathogens or toxins
 4. The methodof claim 1, wherein the different sensors are configured to test fordifferent toxins.
 5. The method of claim 1, wherein the verticallystacked electrodes comprise at least one working electrode, a referenceelectrode and a counter electrode, with each of the working, referenceand counter electrodes positioned one above another and isolated by anelectrical insulator therebetween and having aligned apertures thatdefine at least a part of a fluidic flow channel.
 6. The method of claim5, wherein the working electrode has an inner wall that surrounds anddefines the working electrode aperture, and wherein at least a portionof the working electrode inner wall comprises a predetermined materialanalyte for contacting a sample flowing through a respective fluidicflow channel.
 7. The method of claim 6, wherein the predeterminedmaterial comprises a bioactive material of one or more of the following:an antibody, an antigen, a nucleic acid, a peptide nucleic acid, aligand, a receptor, avidin, biotin, Protein A, Protein G, Protein L, asubstrate for an enzyme and any combination thereof.
 8. The method ofclaim 1, wherein the fluid channels are microfluidic fluid flowchannels.